1. Field of the Invention
The invention relates generally to the field of optical microscopy, and in particular to an automatic microscope scanner enabling rapid three dimensional image acquisition.
2. Related Art
Automated microscope scanners such as disclosed in U.S. Pat. No. 6,711,283, which is incorporated herein in its entirety, enable rapid digitizing of an entire glass slide with a biological sample. These microscope scanners are particularly efficient when the sample thickness, i.e. the depth of the specimen on the glass is smaller or close to the depth of field of the microscope lens. The depth of field is the distance in front of and behind the theoretical focal plane where the specimen appears to be in focus.
A problem commonly arises when the specimen has a thick volumetric structure. On one hand, smaller depth of field provides better spatial resolution along the third dimension of the volumetric specimen and reduces image fusion from adjacent depth layers. On the other hand, it becomes increasingly difficult to ensure focus in microscope scanners with small depth of field as the thick specimen may have a significant depth variance within the field of view. The microscopist may need to observe the same region of the specimen at multiple focal planes. Consequently, such volumetric specimens cannot be digitized efficiently with two-dimensional scanners.
Conventional scanners are designed to ensure a tradeoff between the focus quality and the magnification at which the sample can be viewed. While higher magnification microscope objective lenses normally have higher numerical apertures (NA) and provide the microscopist with higher resolution images, the depth of field decreases. For example, at low magnification such as 10 times (10×) and small NA such as 0.25, a microscope system may have the depth of field of 8.5 um. At higher magnification such as 40× and larger NA such as 0.65, the depth of field is reduced to 1.0 um. At high numerical aperture lenses, depth of field is determined primarily by wave optics, while at lower numerical apertures, the geometrical optical circle of confusion dominates the phenomenon.
One can approach a solution to the depth of field problem by improving the focus accuracy of the microscope scanner. Existing automatic focus methods include such techniques as pre-focusing in points obtained from the macro focus image and generating a three-dimensional data set corresponding to an optimal specimen distance as disclosed in U.S. Pat. No. 6,816,606. Provided the scanner follows precisely this three dimensional profile, it captures an image with increased contrast. Other autofocus methods include tilt designs of either the glass sample or the sensor detecting the focus position. For example, some methods for high speed autofocus by means of tilted designs are described in U.S. Pat. No. 6,677,656 and US Patent Publication Nos. 2005/0089208, 2004/0129858.
The focus accuracy is important at high magnifications to the extent of the specimen thickness. After an accuracy level is achieved matching the thickness of the specimen, no further advance can be made to improve the focus quality of the digital image. In particular, a thick specimen with a three dimensional structure or volume texture cannot be fully represented by its two dimensional image. A linear array sensor typically used in scanning microscopes cannot capture optical images from different focal planes within its field of view at the same time. If the depth of specimen structure varies to a large extent, no uniform focus can be attained within the field of view.
The above focus problem can be addressed by capturing a series of digital images from different focal planes. This series is known as an image stack or an image volume and provides an extended depth of field and preserves the three dimensional structure of the specimen. Image volumes can be further transformed into a three dimensional model or fused into a two dimensional image with enhanced focus. A number of microscope designs are available to acquire image stacks, for example, described in US Patent Publication No. 2004/0264765.
A conventional approach to image stack acquisition is repeated scans with a linear or area scan camera. A drawback of this approach is a substantial increase of the scanning time and memory usage to store digital images. For example, in order to capture a stack of 20 images, a conventional scanner will need to perform 20 runs followed by alignment and stitching procedures. An area scan microscope working in an image stack tiling mode is likely to be inadequately slow and may introduce stitching artifacts to the output volume image. This performance drawback may be critical for express diagnostic applications in a clinical environment and real-time surface inspection applications in an industrial environment.
Therefore, what is needed is a system and method that overcomes these significant problems found in the conventional systems as described above.